Ras molecules are highly conserved signaling elements that mediate cellular responses to a variety of extracellular signals. Ras pathways have been extensively studied in mammalian systems and implicated in the pathogenesis of many human malignancies. Among fungi, Ras elements regulate many differentiation events including yeast-mycelium transitions. The human fungal pathogen Cryptococcus neoformans is a basidiomycete with a well-defined sexual state. The signals that regulate the differentiation events of mating and hyphal development include a pheromone-response pathway, analogous to mammalian MAP kinase pathways. Recently, it has been demonstrated that C. neoformans Ras1 is required for pheromone gene induction and thus for mating. In addition to its role in cellular differentiation, C. neoformans Ras1 also regulates growth at 37 degrees C by signaling pathways distinct from the pheromone-response/MAP kinase pathway. In this proposal, the Alspaugh laboratory will use several unbiased genetic methods to further characterize elements of the Ras pathways of C. neoformans that regulate growth at elevated temperature and mating. First, proteins that physically interact with Ras1 will be identified using a yeast two-hybrid screen. It is expected that genes encoding both upstream and downstream components of the Ras1 signal transduction pathway will be identified by this method. The roles of these proteins will be investigated by targeted gene disruption and genetic epistasis experiments. Additionally, downstream components of Ras proteins that regulate growth at 37 degrees C will be identified by two complementary approaches. Positive effectors of Ras1 signaling will be identified by multicopy suppressor analysis. Inhibitory or regulatory proteins involved in Ras1 control of high temperature growth will be identified using random insertional mutagenesis in the ras1 mutant background and screening for the restoration of growth at 37 degrees C. Finally, in collaboration with other mycology laboratories at Duke University, the Alspaugh lab is helping to construct gene microarrays based on the C. neoformans genome project. Genes that are transcriptionally regulated by Ras1 activity will be identified using these gene microarrays. The relevance of genes identified by any of these approaches will be assessed by gene disruption and analysis of the resulting mutant strain both in vitro as well as in vivo in animal models of cryptococcosis.